Postnatal Development of the Central Nervous System: Anomalies in the Formation of Cerebellum Fissures
Version of Record online: 20 JAN 2010
Copyright © 2010 Wiley-Liss, Inc.
The Anatomical Record
Volume 293, Issue 3, pages 492–501, March 2010
How to Cite
Cerri, S., Piccolini, V. M. and Bernocchi, G. (2010), Postnatal Development of the Central Nervous System: Anomalies in the Formation of Cerebellum Fissures. Anat Rec, 293: 492–501. doi: 10.1002/ar.21082
- Issue online: 17 FEB 2010
- Version of Record online: 20 JAN 2010
- Manuscript Accepted: 14 OCT 2009
- Manuscript Received: 15 JUL 2009
- University of Pavia (Fondo Ateneo Ricerca)
- central nervous system malformations;
- cerebellar cortex;
- growth and development;
A natural defect in rat cerebellum postnatal development has been found in the fissura prima, consisting in various complex configurations of the cerebellar layers. We investigated the genesis of fissure malformations through immunoreactions for PCNA, GFAP, GABAA α6, and calbindin to label proliferating cells of the external granular layer (egl), radial glial fibers, mature granule cells, and Purkinje cells, respectively. Results on critical stages of rat postnatal development provided interesting evidences on how the malformation develops in fissures prima and secunda. Early (postnatal day 10) at the site of malformation, the Bergmann radial glia was often retracted and showed distortions and irregular running. The interruption of GFAP-positive radial glial fibers could fit in with the presence of clusters of PCNA-unlabeled cells in the sites of fusion of the egl; the clusters of cells are granule cells since their soma is labeled by GABAA α6. Moreover, an altered migration of granule cell precursors to the internal granular layer was evident which, in turn, affected Purkinje cell differentiation and the growth of their dendrites. In summary, the changed relationship among glial fiber morphology, granule cell migration, and Purkinje cell differentiation suggests how the Bergmann glial fibers have a basic role in the foliation process, being the driving physical force in directing migration of the granule cells at the base of fissure. Anat Rec, 2010. © 2010 Wiley-Liss, Inc.
The conservation of cerebellum morphology in mammals suggests that foliation patterning is genetically determined (Altman and Bayer, 1997). In fact, several genes have been found to influence the different cells involved in the processes of foliation and fissuration in the mouse and rat cerebellum (Millen et al., 1999). The three-layered cerebellar cortex, composed of the molecular layer (ml), the Purkinje cell layer (Pcl), and the internal granular layer (igl) may be naturally altered. Cerebellar abnormalities have been recently described in neuroradiological literature (Demaerel, 2002) and in articles on adult B6 mice (Tanaka and Marunouchi, 2005) or in rats exposed to neonatal X-irradiation (Li et al., 2006).
A common, natural alteration has been observed in the fissura prima of neonatal rats consisting of various complex configurations of the cerebellar layers extending to the midsagittal plane (Griffin et al., 1980). The first signs of this malformation appeared in the rat at postnatal day 10 (Necchi et al., 2000), beginning with the fusion of the external granular layer (egl) at both sides of the fissura prima, accompanied by an interruption of the pia mater. The involvement of the meninges in the development of the perturbed foliation pattern has been claimed (Stoughton et al., 1978; Lyon et al., 1993; Necchi et al., 2000; Zerbalis et al., 2007).
The molecular mechanisms underlying the spatial and temporal onset of fissure formation (Corrales et al., 2004, 2006) are currently under study in genetic mutants: the levels of sonic hedgehog (Shh), a secreted factor expressed in Purkinje cells from prenatal day 17.5 onwards in the mouse, regulate the extent, but not the pattern, of cerebellar foliation. Shh also regulates the number of folia through its influence on granule cell precursor proliferation.
On the other hand, architectural cerebellar defects were also found to result from a failure of Bergman glia to undergo correct differentiation in the developing cerebellum (Hoser et al., 2007). Studies on human neuronal migration disorders demonstrate how defects in migration as well as cell proliferation, survival, and differentiation may contribute to the malformation genesis (Ross and Walsh, 2001). In particular, it must be considered that the form and circuitry of the cerebellum develop by a complex process that requires integration of afferent-target interactions between multiple neuronal populations and migratory patterns established by neuron-glia interactions.
To explain the main morphological events of formation of fissure defects, we labeled the fundamental cells that might be involved in the growth and differentiation, and establishment of cerebellar cortex architecture, i.e., granule cell precursors, Bergmann glial radial fibers, mature granule cells, and Purkinje cells. We evaluated the pattern/interaction of these cell populations in the following critical developmental stages: at postnatal day 10 (PD 10) and PD 11, when the first sign of malformation begins (Necchi et al., 2000) and active granule cell proliferation occurs (Altman, 1972a; Altman, 1982; Pisu et al., 2005), at PD 17, when granule cell proliferation and migration decline (Altman, 1972a, 1982; Pisu et al., 2005) and PD 30, i.e., after the end of histogenetic processes (Altman, 1982) in the rat.
MATERIALS AND METHODS
Wistar rats were kept in an artificial 12 hr light:12 hr dark cycle and were provided rat chow and tap water ad libitum, throughout the experiment.
The rats at 10 postnatal days (PD10), 11 postnatal days (PD11), and 17 postnatal days (PD17) of age (five per stage) were deeply anesthetized with an intraperitoneal (i.p.) injection of 35% chloral hydrate (100 μL/100 g body wt; Sigma, St. Louis, MO); the brains were quickly removed, fixed in Carnoy solution (6:3:1) for 48 hr, then placed in absolute ethanol and in acetone, and embedded in Paraplast X-tra. Sections (8 μm thick) of the cerebellar vermis, including paravermis, were cut serially in the sagittal plane; one of every six sections was collected on silan-coated slides. The slides were then processed for glial fibrillary acidic protein (GFAP, a marker for glial cells), calbindin (CB, a marker for Purkinje cells), and proliferating cell nuclear antigen (PCNA, a marker for proliferating cells) immunohystochemistry.
Another group of rats at PD11, PD17, and PD30 stages (three per stage) were anesthetized with 35% chloral hydrate and perfused intracardially with saline followed by 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.3. The brains were quickly excised, postfixed in the same fixative medium at 4°C for 1.5 hr, kept in 30% sucrose in 0.1 M phosphate buffer at 4°C for 48 hr and frozen in liquid N2. Twelve micrometer thick cryostatic sections of the cerebellar vermis, including paravermis, were cut serially on the sagittal plane; one of every 10 sections was collected on silan-coated slides. The slides were then processed for GFAP, CB, and GABA A receptor α6 (GABAA α6, a marker for mature cerebellar granule cells) immunohystochemistry.
To avoid possible staining differences due to small changes in the procedure, the reactions were carried out simultaneously on slides from animals at different stages.
All experiments were performed according to the guidelines for care and use of laboratory animals as published by the Italian Ministry of Health (DDL 116/92). All efforts were made to minimize the number of animals used and their suffering.
The paraffin sections were dewaxed in xylene, rehydrated in a decreasing ethanol series, rinsed in distilled water, and immunostained as described below.
The endogenous peroxidases were suppressed by incubation of sections with 3% H2O2 in 10% methanol in phosphate buffered saline (PBS; Sigma, St. Louis, MO) for 7 min. Subsequently, the sections were incubated for 20 min in normal serum at room temperature to block nonspecific antigen binding sites. Localization of PCNA, GFAP, and CB was achieved by applying on brain sections, respectively, mouse monoclonal anti-PCNA (1:600; Oncogene, Boston, MA), goat polyclonal anti-GFAP (1:100; Santa Cruz Biotechnology, Santa Cruz, CA), and rabbit monoclonal anti-CB (1:2000, Sigma, St. Louis, MO) in PBS overnight in a dark moist chamber. Thereafter, the sections were sequentially incubated with biotinylated secondary antibodies (1:200, Vector Laboratories, Burlingame, CA) for 30 min and horseradish peroxidase conjugated avidin-biotin complex (Vector Laboratories, Burlingame, CA) for 30 min at room temperature. Then, 0.05% 3,3′-diaminobenzidine tetrahydrochloride (DAB; Sigma, St. Louis, MO) with 0.01% H2O2 in Tris-HCl buffer (0.05 M, pH7.6) was used as a chromogen. After each reaction step, sections were washed thoroughly in PBS (two changes of 5 min each). Sections were dehydrated in ethanol, cleared in xylene, and mounted in Eukitt (Kindler, Freiburg, Germany). Some sections were counterstained with hematoxylin to facilitate analysis of the results.
The slides were observed with an Olympus BX51 microscope and the images were recorded with an Olympus Camedia C-5050 digital camera and stored on a PC. Corrections to brightness and contrast, as well as the conversion of color images to gray scale images, were made with Paint Shop Pro 7 (Jasc Software).
For control staining, some sections were incubated with PBS instead of the primary antibody. No immunoreactivity was present in these conditions.
In addition to the immunoperoxidase staining, the immunofluorescence staining for GFAP and CB followed by the counterstain with Hoechst 33258 were carried out to find the presence and the distribution of cells in the points of fissure fusions. Moreover, GABAA α6 immunofluorescence followed by the counterstain with Hoechst 33258 was performed to identify the nature of these cells.
Immunoreactions were performed overnight at room temperature on cryostatic sections using a primary goat polyclonal antibody anti-GFAP (1:500; Santa Cruz Biotechnology, Santa Cruz, CA), a mouse monoclonal antibody anti-CB (1:6000; Swant, Bellinzona, Switzerland) and a rabbit polyclonal antibody anti-GABAA α6 receptor (1:1000; Millipore, Billerica, MA) diluted in PBS. Sections were washed in PBS and incubated with the secondary antibodies Alexa-Fluor 594 donkey anti-goat (1:100, Molecular Probes, Space, Milan, Italy), Alexa-Fluor 594 donkey anti-mouse (1:100, Molecular Probes, Space, Milan, Italy), and Alexa-Fluor 488 donkey anti-rabbit (1:100, Molecular Probes, Space, Milan, Italy) in PBS for 1 hr. After washing with PBS, the nuclei were counterstained with 0.1 μg/mL Hoechst 33258 for 6 min, and coverslips were lastly mounted in a drop of Mowiol (Calbiochem, San Diego, CA). Slices were viewed by fluorescence microscopy with an Olympus BX51 equipped with a 100W mercury lamp used under the following conditions: 330–385 nm excitation filter (excf), 400-nm dichroic mirror (dm), and 420-nm barrier filter (bf), for Hoechst 33258; 540 nm excf, 480 nm dm, and 620 nm bf for Alexa 594 and 450–480 nm excf, 500-nm dm, and 515 nm bf for Alexa 488. Images were recorded with an Olympus Camedia C-5050 digital camera and stored on a PC. Images were optimized for color, brightness, and contrast by using Paint Shop Pro 7 software (Jasc Software).
For control staining, some sections were incubated with PBS instead of the primary antibodies. No immunoreactivity was present in these sections.
Cerebellar Malformations at PD 30
At PD 30, histogenetic processes are completed (Altman, 1982). Analysis of sagittal sections of the cerebellar vermis at this time showed evident alterations in the cytoarchitecture of the fissura prima and the fissura secunda. With respect to the normal morphology, there were marked changes in the layering pattern of the cerebellar cortex (Fig. 1A–C). The fluorescence staining for calbindin followed by the counterstain with Hoechst (Fig. 1) and the GABAA α6 immunofluorescence (not shown) evidenced the spatial relation between Purkinje cells and granule cells in the points of fissure malformation. Fusions or islands of the molecular layer bounded by the somata of Purkinje cells, showing intersected terminal branches of the dendrite tree in the ml, were present (Fig. 1D–F); within the islands of molecular layer there were sometimes groups of granule cells (Fig. 1G–I). The pattern of the anomalies varied in the cerebellar sections of the same animal, confirming the great variability of their morphology, not only in the sagittal plane but also in the transversal plane.
Cerebellar Malformations During Histogenesis
The genesis of architectural malformations were studied at some particularly critical stages of postnatal cerebellar morphogenesis (i.e., at PD 10, PD 11, and PD 17) through immunoreactions for PCNA, GFAP, GABAA α6, and CB to label proliferating cells of the egl, radial glial fibers, mature granule cells, and Purkinje cells, respectively.
At PD 10, points of fusion of the two sides of the fissures prima and secunda were detected (Fig. 2A). These zones, located in the deepest parts of fissures, were more visible at PD 11 (Fig. 2B). The PCNA immunoreaction showed that points of fusion are formed by PCNA-labeled cells and also by groups of unlabeled cells intermingled with some labeled cells (Fig. 2C,D). The PCNA-unlabeled cells are, in general, roundish and morphologically similar to granule cells of the igl (Fig. 2C). Elongated, unlabeled cells were also found (Fig. 2D). These cells are likely migrating cells of the egl premigratory zone; in fact, due to the altered morphology of the fissures, the egl could be sectioned in depth. The fluorescence staining for GFAP and the Hoechst counterstain allowed to display the appearance of glial fibers and, at the same time, to detect the presence of nuclei/cells in the points of fusion; while the DAB staining showed better the irregular trend of Bergmann glial fibers. The GABAA α6 immunoreaction labeled the clusters of roundish cells (Fig. 3A–C) within the points of fusion as well as the granule cells of the igl (Fig. 3A,D,E); on the contrary, the elongated cells were not GABAA α6 immunoreactive (not shown).
At these stages, the GFAP-positive radial glial fibers in the egl were interrupted and lacked the endfeet contacts; in the molecular layer, the radial glial fibers were irregular and disorganized (Fig. 4A,B,E,F) as compared with the characteristic, parallel-running features of normal fibers (Fig. 4C,D). In particular, at PD 10, in the points of fusions on the two sides of fissures, one side was characterized by regular Bergmann glial fibers whereas the opposite side had altered glial fibers (Fig. 4A).
Unlike the immunofluorescence, the DAB staining for CB evidenced better the abnormalities in the orientation of Purkinje cell tree in the areas of fissure fusion. The immunoreaction for CB showed that, at PD 10 and PD 11, in the points of fusion, the dendritic trees of the Purkinje neurons were altered in their extension and reoriented, mainly in the distal parts (Fig. 5B–D) [to be compared with a normal picture (Fig. 5A)].
At PD 17, the most interesting finding concerns the apparent continuity of the dendritic tree of Purkinje neurons encircled by the molecular layer islands. As shown by CB immunoreaction, with respect to the normal morphology (Fig. 5E), the dendrite branches of the Purkinje neurons of one side seemed to invade the opposite side (Fig. 5F); this change occurred with reorienting of the distal branches of the dendrite (Fig. 5G) similar to that observed at PD11. Poorly developed dendrites of Purkinje cells were also present (Fig. 5H).
At the same stage, when the proliferating egl is normally reduced to two or three rows of PCNA-positive cells, it was also evident that the malformations progressed along half of the fissures (Fig. 6A,B). Sometimes, at the base of the fissure, an absence of side fusion with thin labeled egl was noticed (Fig. 6A,B,D). Where fusion did occur, interruption of GFAP-labeled glial fibers was evident, as well as the loss of endfeet and the parallel-running trend (Fig. 6E,F). Moreover, there were numerous islands with central molecular layer (Fig. 6B) or igl (Fig. 6C), as described at PD 30 (Fig. 1).
The origin and the primary cause of natural malformations of fissures are still debated. The studies on transgenic mice (Hoser et al., 2007) showing cerebellar anomalies in the foliation process suggest that defects in the formation of folia and fissures are under genetic control. Anomalies have been frequently found over extended breeding cycles for a given breeder (Ezerman and Kromer, 1985; Ramos et al., 2008) as well as in B6 mice, a strain widely used for the generation of knockout mice (Tanaka and Marunouchi, 2005).
As regards the involvement of meninges in the fusion between two opposite folia, there are data supporting that meninges developed and were lost during the histogenetic process (Necchi et al., 2000; Necchi and Scherini, 2002). Several reports have implicated the pial basement membrane as an important player in brain development; in fact, abnormal brain development has been observed after chemical ablation of meningeal cells (Sievers et al., 1994) and after the targeted deletion of basement membrane constituents (Arikawa-Hirasawa et al., 1999; Costell et al., 1999) or deletion of receptors for basement membrane proteins (Graus-Porta et al., 2001). Damages to the pial basement membrane result in changes in radial glial cell morphology. In some mutant mice, the pial basement membrane appeared discontinuous and radial glial cells lost their footing to the pial surface and retracted wherever the pial basement membrane was ruptured (Halfter et al., 2002; Zerbalis et al., 2007).
In a monitoring experimental plan, we demonstrated that the first signs of malformation in Wistar rats were evident in 40% of rats at 10 days of life (Necchi et al., 2000). Starting from this date, we analyzed crucial phases of cerebellum development, during which processes of cell proliferation and maturation, cell migration, and Purkinje cell dendrite growth are differently involved in the patterning of cerebellar architecture.
The results reported here provide interesting evidence on how the malformation develops to produce the grossly disturbed layering of cerebellum (Griffin et al., 1980; Necchi et al., 2000). At PD 10 and PD 11, i.e., in the stages of active granule cell proliferation and migration, the GFAP-positive fibers of the Bergmann radial glia were often retracted due to the loss of endfeet, at least on one side of the fissure. Besides the interruption, the Bergmann glial radial fibers showed distortions and irregular running. Defects of Bergmann glia from loss to relatively minor alterations in their localization or in the organization, length, and endfeet contacts of their processes are reported to affect the cerebellar architecture (Delaney et al., 1996; Kaartinen et al., 2001; Qu and Smith, 2005; Yue et al., 2005; Weller et al., 2006). The severity of the defects correlates with the degree of the disturbed cerebellar layering, in which a misplacement of other neuronal cell types has also been reported. In GFAP-Sox4 transgenic mice, the characteristic layering was not achieved. Indeed fissures and lobules did not form, and neuronal subtypes failed to reach their proper destination and aggregated in clusters (Hoser et al., 2007); in this mutant, radial glia failed to migrate to the normal position and did not extend radial fibers to the pial surface.
Alterations of Bergmann glial radial fibers can affect granule cell migration as observed in methylazoxymethanol (MAM) (Lafarga et al., 1998)—or cisplatin—(Pisu et al., 2005) treated rats or in neonatal X-irradiated rats (Ferguson, 1996; Li et al., 2006), resulting in ectopic displacement of these neurons in the molecular layer. Because radial glial cells are the substrate for neuronal migration (for a review see Rakic and Caviness, 1995), our findings indicate that inward migration of cells from egl to the igl was affected. Consequently, there are two possibilities for the fate of immature cells of the egl: either they can differentiate in loco or they can migrate to displaced areas different from the orthotopic igl. The presence of clusters of PCNA-unlabeled cells at the sites of fusion of the egl, related with the interrupted (or retracted) GFAP-positive radial glial fibers, supports the former possibility. Moreover, the immunolabeling for GABAA α6, i.e., a specific marker for mature granule cells (Thompson and Stephenson, 1994), in the cell clusters of the points of fusion allow us to identify these cells as ectopic granule cells. Nevertheless, we cannot exclude the latter possibility which may be explained by the irregular running of the glial fibers.
On the other hand, altered migration of granule cells affects the growth of Purkinje cell dendrites since the granule cells represent trophic factors for Purkinje cell differentiation (Altman, 1982). In this light, we propose that the abnormal development of Purkinje cell dendrites detected at PD10 and PD11 is likely due to a changed guiding influence of parallel fibers as already observed by Altman (1973) as also may occur after cytotoxic injury (Pisu et al., 2004, 2005). Changes in this relationship may result in the disturbed cerebellar cytoarchitecture.
The changed relationship among glial fiber morphology, granule cell precursor migration, and Purkinje cell differentiation fits very well with the suggestions and the cellular model proposed by Sudarov and Joyner (2007). These authors, using a combination of cell analysis and mutant mice, demonstrated that the crucial events of the initial formation of cerebellar fissures only concern the acquisition of a distinct cytoarchitecture in the “anchoring centers,” i.e., regions that will become the base of each fissure. In the anchoring centers, a basic role is assumed by the granule cells and the Bergmann glial fibers, the latter being involved in the function of the centers by directing migration of the granule cells at the base of fissure. The Purkinje neurons represent a further, important component of the anchoring centers. It has been recently shown (Corrales et al., 2004, 2006) that Shh secreted by Purkinje cells regulates the number of folia via its influence on granule cell precursor proliferation in the egl. The analysis of Engrailed2 (En2) mutants (Sudarov and Joyner, 2007) suggests the existence of a morphogenetic clock that normally controls the timing of the genetic and cellular events that direct formation of the anchoring centers; the loss of En2 disturbs this morphogenetic clock leading to aberrations in the formation of fissures.
In this light, we propose that the timing of formation of the fusion of the fissure sides at PD 10 might be due to a defect in the morphogenetic clock. It is worth noting that the Purkinje cells have a critical differentiation phase (Altman, 1972b; Altman and Bayer, 1985; Pisu et al., 2003) at this stage of cerebellar development which is very crucial phase for the development of lobules VI-VIII, between fissura prima and secunda (Altman, 1982).
Finally, on the grounds of our observations concerning the extension pattern of malformation, it is interesting to note that there could be a link between the gradient of maturation of cortical elements along the lobule (Inouye and Murakami, 1980; Altman and Bayer, 1985, 1997) and the genesis of this anomaly. However, in the study of malformation onset, it might be of interest to also consider the lateromedial gradient of time of cerebellar cell population origin (Martí et al., 2001).
In summary, we conclude that genetic/molecular defects could impede the mechanical forces (granule cells and Bergmann glial fibers) of foliation leading in turn to the altered neuroarchitecture observed in the malformation of cerebellar fissures, in which neural plasticity of cerebellar architecture occurs. The changes in the growth and remodeling of Purkinje cell dendrites are an evident proof of brain plasticity as shown during postnatal histogenesis after X-ray irradiation or treatment with cytotoxic substances (Ferguson, 1996; Avella et al., 2006; Li et al., 2006) or in mutant mice (for a review see Katsuyama and Terashima, 2009).
The authors thank Dr. H. Ladinsky for the discussion and language assistance.
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